Pectin methylesterase (PME) and invertase (INV) are key enzymes in plant carbohydrate
metabolism. An important post-transcriptional mechanism of regulation of these enzymes is represented
by proteinaceous inhibitors. PME inhibitors (PMEI) and INV inhibitors (INH), belong to the same
structural family Pf 04043. Despite the structural similarity, these two inhibitors act on two
structurally and functionally different enzymes, but never both. Both inhibitors do not inhibit fungal
enzymes indicating that they are mainly involved in growth and development even if they seems to be also
involved in defence against pathogens.
In this thesis through a functional genomics approach a pectin methylesterase inhibitor
(SolyPMEI) and an invertase inhibitor (SolyCIF) of Solanum lycopersicum have been identified and
characterised.
SolyPMEI was mainly expressed in red fruits and, albeit at lower levels, in flowers and pollen. The
SolyPMEI cDNA was cloned and expressed in Pichia pastoris. All attempts to produce active
recombinant SolyPMEI protein appeared to be unsuccessful, neverthless CD spectra and disulfide bridges
indicated a correct folding of the protein in vitro. The immuno-affinity fishing approach was used to purify
the natural SolyPMEI from tomato fruits. The isolation of both natural SolyPMEI and PME-1 indicated
that SolyPMEIs is in vivo engaged in the formation of a stable complex with endogenous PMEs.
SolyCIF was mainly expressed in leaves, flowers and green fruits of the plant and localized in the
cell wall compartment. The SolyCIF cDNA was cloned and expressed in Pichia pastoris. The purified
recombinant protein was biochemically characterized. The invertase activity was strongly inhibited in a
dose-dependent manner by recombinant SolyCIF.With an affinity chromatography approach, the natural
ligand of the inhibitor (the vacuolar invertase namely TIV-1) was purified and characterised. TIV-1 has
been shown to be naturally proteolyzed and it was established that the fragments produced have to be
tightly associated for its enzymatic activity to occur. N- terminal sequencing of fragments and the
molecular model of TIV-1 shows that the fragmentation splits the catalytic site of the enzyme into two
halves, which confirms that the enzymatic activity is possible only when the fragments are tightly
associated....more